Composition for diagnosing liver cancer and methods of diagnosing liver cancer and obtaining information for diagnosing liver cancer

ABSTRACT

Provided is method of diagnosing liver cancer in a subject, the method comprising contacting a sample from a subject with a substance that specifically binds to transmembrane emp24 domain trafficking protein 2 (TMED2), cluster of differentiation 43 (CD43), or any combination thereof on the surface of a microvesicle; and measuring the level of the substance bound to microvesicles in the sample; and related methods and compositions.

RELATED APPLICATION

This application claims the benefit of Korean Patent Application No.10-2013-0122962, filed on Oct. 15, 2013 in the Korean IntellectualProperty Office, the entire disclosure of which is hereby incorporatedby reference.

INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: 17,898 bytes ASCII (Text) file named “716092_ST25.TXT,”created Jun. 30, 2014.

BACKGROUND OF THE INVENTION

1. Field

The present disclosure relates to compositions for diagnosing livercancer in a subject and methods of diagnosing liver cancer and obtaininginformation for diagnosing liver cancer in a subject.

2. Description of the Related Art

When it is difficult or impracticable to perform a biopsy to diagnose adisease such as cancer, a diagnostic method using biological fluids suchas blood, urine, or saliva may be employed. However, due to the absenceof a high accuracy marker, there are difficulties in diagnosing diseasessuch as cancer using biological fluids. In the case of diagnosing livercancer, alpha-fetoprotein (AFP) is a widely known plasma protein thatcan be used as a marker, but AFP has a low sensitivity for detection ofliver cancer. In addition, AFP levels are increased in patients withcirrhosis as well. Accordingly, it is difficult to use patients' AFPlevels to detect progression from cirrhosis to liver cancer.

When a biopsy is used to diagnose cancer, a patient may suffer from aninvasive procedure such as an incision. Also, when an error occursduring sampling of a specimen for examination, the diagnostic accuracymay be low.

Transmembrane emp24 domain trafficking protein 2 (TMED2) is a proteinencoded in humans by the TMED2 gene. Research indicates that TMED2specifically binds to Golgi reassembly-stacking protein 1 (GORASP1) andGolgi reassembly-stacking protein 1 (GORASP2).

Cluster of differentiation 43 (CD43), which is also known as sialophorin(SPN) or leukosialin, is a transmembrane cell surface protein encoded inhumans by the SPN gene. CD43 is a major sialoglycoprotein on the surfaceof human T lymphocytes, monocytes, granulocytes, and some B lymphocytes,wherein CD43 appears to be important for the immune function. CD 43 maybe a part of a physiologic ligand-receptor complex involved in T-cellactivation.

However, there is no evidence that TMED2 and/or CD43 is associated withliver cancer.

BRIEF SUMMARY OF THE INVENTION

A method of diagnosing liver cancer in a subject, the method comprisingcontacting a sample from a subject with a substance that specificallybinds to transmembrane emp24 domain trafficking protein 2 (TMED2),cluster of differentiation 43 (CD43), or any combination thereof on thesurface of a microvesicle; measuring the level of the substance bound tomicrovesicles in the sample; measuring the level of the substance boundto microvesicles in the control sample; and determining that the subjecthas a liver cancer or an increased likelihood of developing a livercancer when the level of the substance bound to microvesicles in thesample is equal to or greater than a threshold level, or when the levelof the substance bound to microvesicles in the sample is greater thanthe highest level among levels obtained from the plurality of samplesobtained from cirrhosis patients, wherein the control sample includes aplurality of samples obtained from liver cancer patients and a pluralityof samples obtained from cirrhosis patients, and the threshold level isa value such that the probability of a sample to be a liver cancerpatient is equal to or greater than 50% to 100% and the probability of asample to be a liver cancer patient is obtained by analyzing themeasured level with a statistic model. Other methods and relatedcompositions are also provided.

Additional aspects will be set forth in part in the description whichfollows and, in part, will be apparent from the description, or may belearned by practice of the presented embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

These and/or other aspects will become apparent and more readilyappreciated from the following description of the embodiments, taken inconjunction with the accompanying drawings in which:

FIG. 1 is a graph showing a receiver operating characteristic (ROC)curve for determining the presence of cirrhosis or liver cancer in asample based on band intensity values of protein measured inmicrovesicles that are separated by using beads coated withanti-transmembrane emp24 domain trafficking protein 2 (TMED2);

FIG. 2 is a graph showing a ROC curve for determining the presence ofcirrhosis or liver cancer in a sample based on band intensity values ofprotein measured in microvesicles that are separated by using beadscoated with anti-cluster of differentiation 43 (CD43) antibodies;

FIG. 3 is a graph showing a ROC curve for determining the presence ofcirrhosis or liver cancer in a sample based on band intensity values ofprotein measured in microvesicles that are separated by using beadscoated with anti-TMED2 antibodies and beads coated with anti-CD43antibodies; and

FIG. 4 is a graph showing a ROC curve for determining the presence ofcirrhosis or liver cancer in a sample based on the values of mir-210 andmir-346 with respect to the microvesicles that are separated by usingbeads coated with anti-TMED2 and beads coated with anti-CD43,respectively.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings, wherein like referencenumerals refer to like elements throughout. In this regard, the presentembodiments may have different forms and should not be construed asbeing limited to the descriptions set forth herein. Accordingly, theembodiments are merely described below, by referring to the figures, toexplain aspects of the present description. As used herein, the term“and/or” includes any and all combinations of one or more of theassociated listed items.

According to one aspect of the present disclosure, provided is acomposition for diagnosing liver cancer in a subject, wherein thecomposition includes a substance that specifically binds totransmembrane emp24 domain trafficking protein 2 (TMED2), cluster ofdifferentiation 43 (CD43), or a combination thereof.

TMED2 is a protein encoded in human by the TMED2 gene. Researchindicates that TMED2 specifically binds to Golgi reassembly-stackingprotein 1 (GORASP1) and Golgi reassembly-stacking protein 2 (GORASP2).TMED2 may have an amino acid sequence of NP_(—)006806 (SEQ ID NO: 1) andbe encoded by a nucleotide sequence of NM_(—)006815 (SEQ ID NO: 2).

CD43, which is also known as sialophorin (SPN) or leukosialin, is atransmembrane cell surface protein that in human is encoded by SPN gene.CD43 is a major sialoglycoprotein on the surface of human T lymphocytes,monocytes, granulocytes, and some B lymphocytes, wherein CD43 appears tobe important for immune function. CD43 may be a part of a physiologicligand-receptor complex involved in T-cell activation. CD43 may have anamino acid sequence of NP_(—)001025459 (SEQ ID NO: 3) and be encoded bya nucleotide sequence of NM_(—)001030288 (SEQ ID NO: 4).

The TMED2, CD43, or combination thereof may be present in microvesicles,for example, on the surface of the microvesicles derived from a livingbody. The TMED2, CD43, or combination thereof may be presentspecifically on the surface of microvesicles that are separated from asubject with liver cancer. The TMED2, CD43, or combination thereof maybe present at higher amounts on the surface of the microvesicles thatare separated from the subject with liver cancer than on the surface ofmicrovesicles that are separated from a control group sample. Thecontrol group sample may comprise samples from one or more normalsubjects, one or more subjects with a disease other than liver cancer,for example, cirrhosis, or a combination thereof. The control sample mayinclude a plurality of samples obtained from liver cancer patients and aplurality of samples obtained from cirrhosis patients. In this regard,the composition may be used to distinguish the subject with liver cancerfrom the control group, for example subjects with cirrhosis. A positivecontrol also can be used (e.g., the level of TMED2, CD43, or both insamples from one or more known positive liver cancer subjects), in whichcase a lower level of the biomarkers can indicate that the test subjectdoes not have liver cancer. Furthermore, the control (the level ofTMED2, CD43, or both in a sample from a known non-cancerous subject or aknown cancerous subject) can be provided in the form of empiricalmeasurements from such known samples taken before, during, or aftermeasurements of the test sample, or the control can be provided bypre-determined levels of TMED2, CD43, or both (e.g., levels from a knowncancerous or non-cancerous sample or average level of multiple suchsamples previously determined and stored in any form, such as acomparison scale generated from such measurements or data in anon-transitory storage medium.

The microvesicles may be separated from the subject with liver cancer orthe subject with cirrhosis according to methods known in the art. Themethods may include a process of centrifuging the sample, a process offiltering the sample, a process of incubating the microvesicles with asubstance that specifically binds thereto or that is intercalatedbetween a lipid bilayer, or any combination thereof. The incubation maybe carried out in vitro. In addition, examples of the separation methodsof the microvesicles include a process using a solid support orcentrifugal forces, density gradient centrifugation,ultracentrifugation, filtration, dialysis, immunoaffinitychromatography, electrophoresis, or any combination thereof. Thesubstance that specifically binds to the microvesicles may be asubstance capable of binding to surface proteins, lipids, or sugars ofthe microvesicles. Examples of the surface proteins of the microvesiclesinclude CD63, CD83, CD9, integrin-beta 1 (ITGB1), EpCAM, caveolin, FasL,HLA-DRA, CD36, CD63, CD81, MUC1, ERBB4, GPER, ERBB2, MLANA, AMHR2, orany combination thereof. The substance that specifically binds to themicrovesicles may be a substance having binding affinity to proteins,such as an enzyme substrate, a coenzyme, a regulatory factor, or asubstance that specifically binds to receptors, such as lectin, a sugar,a glycoprotein, an antigen, an antibody or a antigen-binding fragmentthereof such as an Fab fragment, a hormone, a neurotransmitter, aphospholipid-binding protein, a pleckstrin homology (PH)domain-containing protein, a cholesterol-containing protein, or anycombination thereof. The Fab fragment comprises antigen-binding sites,and examples of the Fab fragment include a single-domain antibody, aFab, a Fab′, and a scFv. The substance that is intercalated between thelipid bilayer may include a lipophilic moiety, an amphipathic moiety, amoiety of zwitterions, or any combination thereof. Examples of thelipophilic moiety include fatty acid, sterol, or glyceride. Examples ofthe amphipathic moiety include phospholipid or sphingolipid. Examples ofthe moiety of zwitterions include sulfobetaine, carboxybetaine, orphosphoryl choline. The substance that specifically binds to themicrovesicles or that is intercalated between the lipid bilayer may bindto a solid support. The solid support may have a shape of a sphere,polygon, plate, a linear shape, or any combination thereof. The solidsupport may be formed of polystyrene, polypropylene, magnetic particles,or any combination thereof.

The sample may be biological materials derived from the subject. Suchbiological material may include a solid tissue obtained from a fresh orpreserved organ or a tissue sample, or by a biopsy; blood or bloodcomponents; amniotic fluid, peritoneal fluid; bodily fluid such asinterstitial fluid; cells; or any combination thereof. The sample mayinclude compounds, such as a preservative, an anti-coagulant, a buffer,a fixative, a nutrient, and antibiotics, wherein the compounds are notnaturally mixed with the biological material. Examples of the biologicalsample include urine, mucus, saliva, tears, blood, plasma, serum,sputum, spinal fluid, pleural effusion, nipple aspirates, lymph fluid,airway fluid, intestinal fluid, urogenital duct fluid, breast milk,semen, cerebrospinal fluid, bodily fluid of organ system, peritonealfluid, bodily fluid of cystic tumor, amniotic fluid, or any combinationthereof. The sample may also include circulating tumor cells (CTCs). Thesubstance that specifically binds to TMED2, CD43, or combination thereofmay also specifically bind to TMED2, CD43, or combination thereof on thesurface of the microvesicles that are separated from the living body.

The substance that specifically binds to TMED2, CD43, or combinationthereof may be a material that naturally or non-naturally binds toTMED2, CD43, or combination thereof. The substance that may naturallybind to TMED2, CD43, or combination thereof may be, for example, aprotein that specifically binds to TMED2, CD43, or combination thereofwithin the living body. Examples of the substance that specificallybinds to the TMED2, CD43, or combination thereof include an antibody ora antigen binding fragment thereof, a ligand, a substrate, an inhibitor,an agonist, an antagonist, a co-factor, or any combination thereof. Theantibody may be a monoclonal antibody or a polyclonal antibody. Theantigen binding fragment of the antibody may be a single-domainantibody, a Fab, a Fab′, a scFv, or any combination thereof.

The substance that specifically binds to TMED2, CD43, or combinationthereof may be attached to a detectable label. The detectable label maybe primary labels where the label comprises an element that is detecteddirectly or that produces a directly detectable label, or secondarylabels where the detected label binds to a primary label, as is commonin immunological labeling. The detectable label may include an opticallabel, an electrical label, a radioactive label, an enzyme label, or anycombination thereof. The enzyme may be an enzyme converting a substrateto directly detectable labels. The enzyme may be a horse radishperoxidase, alkaline phosphatase, luciferase, etc. The secondary labelsmay include biotin, streptavidin, digoxigenin, primary antiboidies etc.The optical label may be a fluorescent substance or a phosphorescentsubstance. Examples of the fluorescent substance include fluorescein,rhodamine, cyanine (Cy), metalloporphyrin complex, Cy-5, and Cy-3.Examples of the fluorescein dye include 6-carboxyfluorescein (6-FAM),1,2′,4′,1,4,-tetrachlorofluorescein (TET) 2 and2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX) 3,2′,7′dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE)4,2′-chloro-5′-fluoro-7′,8′-fusedphenyl-1,4-dichloro-6-carboxyfluorescein 5, and2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein 6.

The substance that specifically binds to TMED2, the CD43, or acombination thereof may be fixed to a solid support. The solid supportmay be formed of nanoparticles or microparticles. The solid support maybe formed of magnetic particles or non-magnetic particles. The solidsupport may have a shape of a bead, a sphere, a polygon, a plate, or anycombination thereof. The solid support may be in the form of an array towhich the substance that specifically binds to TMED2, CD43, orcombination thereof is fixed in a certain area.

The composition may further include a reagent to measure expressionlevels of micro RNA (mir)-210, mir-346, or a combination thereof. Thereagent may specifically bind to mir-210, mir-346, or a combinationthereof, or may bind to nucleotide sequences each complementary tomir-210, mir-346, or a combination thereof. The reagent may be a naturalsubstance, a synthetic substance, or a semi-synthetic substance. Thereagent may include nucleic acid, for example, DNA, RNA, DNA-RNA hybrid,PNA, or any combination thereof. The nucleic acid may be single, doubleor multi stranded. The nucleic acid may include 10 nucleotides (nt) ormore in length. For example, the nucleic acid may include about 10 nt toabout 1,000 nt, about 10 nt to about 500 nt, about 10 nt to about 100nt, about 10 nt to about 50 nt, about 10 nt to about 30 nt, about 10 ntto about 20 nt, about 15 nt to about 100 nt, about 15 nt to about 50 nt,about 15 nt to about 30 nt, about 15 nt to about 20 nt, about 20 nt toabout 1,000 nt, about 20 nt to about 500 nt, about 20 nt to about 100nt, about 20 nt to about 50 nt, about 20 nt to about 30 nt, about 30 ntto about 1,000 nt, about 30 nt to about 500 nt, about 30 nt to about 100nt, about 30 nt to about 50 nt, about 40 nt to about 100 nt, about 40 ntto about 80 nt, or about 40 nt to about 60 nt in length.

The reagent may include a primer, a probe or an antisense sequencecontaining a sequence specific to mir-210 or a complementary sequencethereof. The reagent may also include a primer, a probe or an antisensesequence containing a sequence specific to mir-346 or a complementarysequence thereof. The reagent may also include any combination ofprimers, probes or antisense sequences containing a sequence specific orcomplementary to mir-210 or mir-346. The primer may serve as apolymerization start site for a polymerase in a polymerase chainreaction (PCR). The primer may be used in a nucleic acid amplificationreaction. The term “amplification” used herein refers to a method ofamplifying copies of a target sequence or a complementary sequencethereof. The nucleic acid amplification reaction may be carried out byany method known in the art. The nucleic acid may be amplified throughmultiple cycles during the amplification reaction, or may be amplifiedin a single temperature or temperature range with a cycle. Examples ofcycling techniques used in the nucleic acid amplification reactioninclude, for example, a method relying on a thermal cycling. The methodusing the thermal cycling may include PCR. PCR is widely known in theart and includes a process of denaturation in which double-stranded DANis denatured into single-stranded DNA by thermal denaturation; a processof annealing in which a primer is annealed to the single-stranded DNA;and a process of elongation in which a new DNA strand complementary tothe single-stranded DNA is synthesized and elongated from the primer. Amethod of isothermal amplification of nucleic acid is a method carriedout in a single temperature or temperature range without a cycle, or isa method relying on main aspects of the amplification process in asingle temperature. In contrast to the PCR in which a reaction productis heated to bind to an additional primer for the separation ofdouble-stranded DNA, the method of isothermal amplification of nucleicacid involves a strand displacing polymerase to separate double-strandedDNA and re-copy a template single-stranded DNA. In order to initiatereiterative template copying, the method of isothermal amplification ofnucleic acid may be divided into a method relying on the stranddisplacing polymerase and the other method relaying on continuous reuseor new synthesis of a single primer molecule. The strand displacingpolymerase-dependent method may be selected from the group consisting ofhelicase dependant amplification (HAD), exonuclease dependantamplification, recombinase polymerase amplification (RPA), and loopmediated amplification (LAMP). The method relying on continuous reuse ornew synthesis of the single primer molecule may be selected from thegroup consisting of strand displacement amplification (SDA) and nucleicacid based amplification (NASBA and TMA). The primer may include one orat least two primer sets according to the selected method. The primermay be a primer used for the PCR.

Mir-210 is a mature microRNA having a nucleotide sequence of SEQ ID NO:5, and mir-346 is a mature microRNA having a nucleotide sequence of SEQID NO: 6.

The reagent may be attached to a detectable label. The detectable labelis defined as described above. The reagent may be fixed to glass or asolid support. The solid support is defined as described above.

The composition may be in any phase. That is, the composition may be ina liquid phase, a solid phase, or a combination thereof.

The subject may be a mammal, and the mammal may be a human, a mouse, acow, a pig, a horse, a sheep, a dog, a cat, or any combination thereof.

The composition described above may be used for the performance of themethod described hereinafter.

According to another aspect of the present invention, there is provideda kit for diagnosing liver cancer in a subject by using the compositionthat includes the substance specifically binding to TMED2, CD43, orcombination thereof.

The substance that specifically binds to TMED2, CD43, or combinationthereof is defined as described above. A kit may further include areagent for diagnosing liver cancer in a subject, and the reagent mayinclude a buffer, an indicator, or a combination thereof. The kitdescribed above may be used for the performance of the method describedhereinafter.

According to another aspect of the present invention, there is provideda method of diagnosing liver cancer in a subject, the method includingcontacting a sample from the subject to a substance that specificallybinds to TMED2, CD43, or combination thereof on the surface ofmicrovesicles in the sample; measuring the level of the substance boundto microvesicles in the sample; measuring the level of the substancebound to microvesicles in the control sample; and determining that thesubject has a liver cancer or an increased likelihood of developing aliver cancer when the level of the substance bound to microvesicles inthe sample is equal to or greater than a threshold level, or when thelevel of the substance bound to microvesicles in the sample is greaterthan the highest level among levels obtained from the plurality ofsamples obtained from cirrhosis patients, wherein the control sampleincludes a plurality of samples obtained from liver cancer patients anda plurality of samples obtained from cirrhosis patients, and thethreshold level is a value such that the probability of a sample to be aliver cancer patient is equal to or greater than 50% to 100% and theprobability of a sample to be a liver cancer patient is obtained byanalyzing the measured level with a statistic model. The threshold levelmay be a value such that the probability of a sample to be a livercancer patient is equal to or greater than 50% to 100%, for example, 50%to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%,80% to 100%, 90% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to90%, 70% to 80%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

The contacting may be carried out in a liquid medium. The liquid mediummay include a liquid sample itself, water, a buffer, or any combinationthereof. The contacting may be carried out by mixing the sample with thesubstance. In some embodiments, the contacting may be carried out bystirring the mixture containing the sample and the substance, whereinthe stirring may be carried out to a certain extent that does notdestroy microvesicles contained therein.

The subject may be a mammal, and the mammal may be a primate. Additionalexamples of the mammal include a human, a mouse, a cow, a pig, a horse,a sheep, a dog, a cat, or any combination thereof.

The sample may comprise biological materials derived from the subject.The biological material may include solid tissue obtained from a freshor preserved organ or tissue sample, or by a biopsy; blood or bloodcomponents; amniotic fluid, peritoneal fluid; bodily fluid such asinterstitial fluid; cells; or any combination thereof. The sample mayinclude compounds, such as a preservative, an anti-coagulant, a buffer,a fixative, a nutrient, and antibiotics, wherein the compounds are notnaturally mixed with the biological material. Examples of the biologicalsample include urine, mucus, saliva, tears, blood, plasma, serum,sputum, spinal fluid, pleural effusion, nipple aspirates, lymph fluid,airway fluid, intestinal fluid, urogenital duct fluid, breast milk,semen, cerebrospinal fluid, bodily fluid of organ system, peritonealfluid, bodily fluid of cystic tumor, amniotic fluid, or any combinationthereof. The sample may also include CTCs.

The TMED2, CD43, or combination thereof may be associated withmicrovesicles, for example, on the surface of the microvesicles derivedfrom the living body. The TMED2, CD43, or combination thereof may bepresent specifically on the surface of microvesicles that are separatedfrom the subject with liver cancer. The TMED2, the CD43, or combinationthereof may be present at higher levels (e.g., higher amounts) in themicrovesicles that are separated from the subject with liver cancer thanin microvesicles that are separated from the control group. The controlgroup sample may be derived from one or more normal, non-caneroussubjects, one or more subjects with a disease other than liver cancer,for example, cirrhosis, or a combination thereof. In this regard, thecomposition may be used to distinguish the subject with liver cancerfrom normal subjects or subjects with cirrhosis. A positive control alsocan be used (e.g., the level of TMED2, CD43, or both in samples from oneor more known positive liver cancer subjects), in which case a lowerlevel of the biomarkers can indicate that the test subject does not haveliver cancer. Furthermore, the control (the level of TMED2, CD43, orboth in a sample from a known non-cancerous subject or a known canceroussubject) can be provided in the form of empirical measurements from suchknown samples taken before, during, or after measurements of the testsample, or the control can be provided by pre-determined levels ofTMED2, CD43, or both (e.g., levels from a known cancerous ornon-cancerous sample or average level of multiple such samplespreviously determined and stored in any form, such as a comparison scalegenerated from such measurements or data in a non-transitory storagemedium.

The substance that specifically binds to TMED2, CD43, or combinationthereof may be a natural or non-natural (synthetic) material taht bindsto TMED2, CD43, or combination thereof. The natural substance that maybind to TMED2, CD43, or combination thereof may be, for example, aprotein that specifically binds to TMED2, CD43, or combination thereofwithin the living body. Examples of the substance that specificallybinds to TMED2, CD43, or combination thereof include an antibody or aantigen binding fragment thereof, a ligand, a substrate, an inhibitor,an agonist, an antagonist, a co-factor, or any combination thereof. Theantibody may be a monoclonal antibody or a polyclonal antibody. Theantigen binding fragment of the antibody may be a single-domainantibody, a Fab, a Fab′, a scFv, or any combination thereof. Theanti-TMED2 antibody may be TMED2 (A-8) (Cat.no. sc-376033: Santa CruzBiotechnology, Inc.), TMED2 (C-8) (Cat.no. sc-376459: Santa CruzBiotechnology, Inc.), TMED2 (H-108) (Cat.no. sc-292002: Santa CruzBiotechnology, Inc.), or anti-TMED2 antibody (ab97600: Abcam®). Theanti-CD43 antibody may be mouse anti-CD43 antibody (Cat.no. LS-B3775,LS-B2792, LS-C134435, or LS-C87781: Lifesapn Biosciences), mouseanti-CD43 antibody (Cat.no. 10-220-C100, 1F-220-T100, 1P-220-T100, orA4-220-T100: EXBIO Antibodies), mouse anti-CD43 antibody (Cat.no.ABIN351514, ABIN260269, ABIN180650, or ABIN96680:antibodies-online.com), or anti-CD43 monoclonal antibody (Cat. No.:FAB2038P: R&D Systems, Inc.).

The substance that specifically binds to TMED2, CD43, or combinationthereof may be attached to a detectable label. The detectable label maybe primary labels where the label comprises an element that is detecteddirectly or that produces a directly detectable label, or secondarylabels where the detected label binds to a primary label, as is commonin immunological labeling. The detectable label may include an opticallabel, an electrical label, a radioactive label, an enzyme label, or anycombination thereof. The enzyme may be an enzyme converting a substrateto directly detectable labels. The enzyme may be a horse radishperoxidase, alkaline phosphatase, luciferase, etc. The secondary labelsmay include biotin, streptavidin, digoxigenin, primary antiboidies etc.The optical label may be a fluorescent substance or a phosphorescentsubstance. Examples of the fluorescent substance include fluorescein,rhodamine, cyanine (Cy), metalloporphyrin complex, Cy-5, and Cy-3.Examples of the fluorescein dye include 6-carboxyfluorescein (6-FAM),1,2′,4′,1,4,-tetrachlorofluorescein (TET) 2 and2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX) 3,2′,7′dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE)4,2′-chloro-5′-fluoro-7′,8′-fusedphenyl-1,4-dichloro-6-carboxyfluorescein 5, and2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein 6.

The substance that specifically binds to TMED2, CD43, or a combinationthereof may be fixed to a solid support. The solid support may be formedof nanoparticles or microparticles. The solid support may be formed ofmagnetic particles or non-magnetic particles. The solid support may havea shape of a bead, a sphere, a polygon, a plate, or any combinationthereof. The solid support may be in the form of an array to which thesubstance that specifically binds to the TMED2, CD43, or a combinationthereof is fixed in a certain area.

The method also includes the measuring of the levels of the substancebound to the microvesicles in the sample and control sample. Themeasuring may be carried out by determining the presence of thesubstance or measuring an amount of the substance when the substance isbound to the microvesicles. The substance may be attached to adetectable label, and accordingly measurement may be carried out bymeasuring the amount of the substance based on signals generated fromthe label. The measuring may be also carried out by determining thepresence of the substance or measuring the amount of the substance,after the substance is separated from the composite (i.e., product)containing the substance and the microvesicles.

The substance that specifically binds to the TMED2, CD43, or combinationthereof may be attached to a detectable label. The detectable label mayinclude an optical label, an electrical label, a radioactive label, anenzyme label, or any combination thereof. The optical label may be afluorescent substance or a phosphorescent substance. Examples of thefluorescent substance include fluorescein, rhodamine, cyanine (Cy),metalloporphyrin complex, Cy-5, and Cy-3. Examples of the fluoresceindye include 6-carboxyfluorescein (6-FAM),1,2′,4′,1,4,-tetrachlorofluorescein (TET) 2 and2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX) 3,2′,7′dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE)4,2′-chloro-5′-fluoro-7′,8′-fusedphenyl-1,4-dichloro-6-carboxyfluorescein 5, and2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein 6.

The measuring of the levels of the substance may include a process ofmeasuring directly an amount of the substance that is separated from themicrovesicles, or a process of measuring indirectly an amount of thesubstance without separating it from the microvesicles. The measuringmay be carried out by detecting signals generated from the detectablelabel labeled to the substance. Means of separating the substance mayinclude centrifugation, precipitation, salting out, dialysis,filtration, chromatography, or any combination thereof. Thechromatography may include affinity chromatography, size-exclusionchromatography, ion exchange chromatography, or any combination thereof.The measuring may be carried out by ELISA, western blotting,electrophoresis, mass spectrometry, spectrometer, or any combinationthereof.

The method may further include, after the contacting the sample with thesubstance that specifically binds TMED2 or CD43 (or both), a process ofseparating the microvesicles from the sample. During the process ofseparating the microvesicles, the microvesicles specifically bound tothe substance that specifically binds to the TMED2, CD43, or combinationthereof are separated from the rest of the sample. The separating of themicrovesicles may be carried out by using a material such as an antibodythat specifically binds to the substance that specifically binds toTMED2, CD43, or a combination thereof. The substance-specific materialsuch as an antibody may be fixed to a solid support, for example,magnetic particles or a plate. Separation of the microvesicles mayinclude a process of contacting the composite, containing themicrovesicles and the substance, to the material, fixed to the solidsupport, that is specifically bound with the substance specificallybinding to TMED2, CD43, or combination thereof.

Separation of the microvesicles may include a process of centrifugingthe sample, a process of filtering the sample, a process of incubatingthe microvesicles with a substance that specifically binds thereto orthat is intercalated between a lipid bilayer, or any combinationthereof. The incubation may be carried out in vitro. In addition,examples of the separation methods of the microvesicles include aprocess using a solid support or centrifugal forces, density gradientcentrifugation, ultracentrifugation, filtration, dialysis,immunoaffinity chromatography, electrophoresis such as free flowelectrophoresis, or any combination thereof. The substance thatspecifically binds to the microvesicles may be a substance capable ofbinding to surface proteins, lipids, or sugars of the microvesicles.Examples of the surface proteins of the microvesicles include CD63,CD83, CD9, integrin-beta 1 (ITGB1), EpCAM, caveolin, FasL, HLA-DRA,CD36, CD63, CD81, MUC1, ERBB4, GPER, ERBB2, MLANA, AMHR2, or anycombination thereof. The substance that specifically binds to themicrovesicles may be a substance having binding affinity to proteins, anenzyme substrate, a coenzyme, a regulatory factor, and a substance thatspecifically binds to receptor, lectin, sugar, a glycoprotein, anantigen, an antibody or a antigen-binding fragment thereof, a hormone, aneurotransmitter, a phospholipid-binding protein, a pleckstrin homology(PH) domain-containing protein, a cholesterol-containing protein, or anycombination thereof. The antigen binding fragment has antigen-bindingsites, and examples of the antigen binding fragment include asingle-domain antibody, a Fab, a Fab′, and a scFv. The substance that isintercalated between the lipid bilayer may include a lipophilic moiety,an amphipathic moiety, a moiety of zwitterions, or any combinationthereof. Examples of the lipophilic moiety include fatty acid, sterol,or glyceride. Examples of the amphipathic moiety include phospholipid orsphingolipid. Examples of the moiety of zwitterions includesulfobetaine, carboxybetaine, or phosphoryl choline. The substance thatspecifically binds to the microvesicles or that is intercalated betweenthe lipid bilayer may bind to a solid support. The solid support mayhave a shape of sphere, polygon, plate, linear, or any combinationthereof. The solid support may be formed of polystyrene, polypropylene,magnetic particles, or any combination thereof.

The method may further include a process of separating the microvesiclesfrom the sample that is separated from the subject, before contactingwith the substance that specifically binds to TMED2, CD43. In this step,the substance that specifically binds to TMED2, CD43, or combinationthereof may not be used. Accordingly, the substance that specificallybinds to TMED2, CD43, or combination thereof may enable to measure anamount of the microvesicles that are present on the surface with respectto the whole microvesicles. The separation of the microvesicles isdefined as described above.

The method includes determining that the subject has a liver cancer oran increased likelihood of developing a liver cancer when the level ofthe substance bound to microvesicles in the sample is equal to orgreater than a threshold level, or when the level of the substance boundto microvesicles in the sample is greater than the highest level amonglevels obtained from the plurality of samples obtained from cirrhosispatients, wherein the control sample includes a plurality of samplesobtained from liver cancer patients and a plurality of samples obtainedfrom cirrhosis patients, and the threshold level is a value such thatthe probability of a sample to be a liver cancer patient is equal to orgreater than 50% to 100% and the probability of a sample to be a livercancer patient is obtained by analyzing the measured level with astatistic model. The statistic model may be linear regression model, alogistic regression model, etc. The threshold level may be a value suchthat the probability of a sample to be a liver cancer patient is equalto or greater than 50% to 100%, for example, 50% to 90%, 50% to 80%, 50%to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%,60% to 90%, 60% to 80%, 60% to 70%, 70% to 90%, 70% to 80%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The probability of a sampleto be a liver cancer patient may be determined by a linear predictorfunction derived from a logistic regression model. The basic idea oflogistic regression is to use the mechanism already developed for linearregression by modeling the probability pi using a linear predictorfunction, i.e., a linear combination of the explanatory variables and aset of regression coefficients that are specific to the model at handbut the same for all trials. The linear predictor function f(i) for aparticular data point I is written as:

f(i)=β₀+β₁χ_(1,i)+ . . . +β_(m)χ_(m,i),

where β₀, . . . , β_(m) are regression coefficients indicating therelative effect of a particular explanatory variable on the outcome.

The linear predictor function may be obtained by using, for example, across-validation method. Cross-validation, sometimes called rotationestimation, is a model validation technique for assessing how theresults of a statistical analysis will generalize to an independent dataset. It is mainly used in settings where the goal is prediction, and onewants to estimate how accurately a predictive model will perform inpractice. It is worth highlighting that in a prediction problem, a modelis usually given a dataset of known data on which training is run(training dataset), and a dataset of unknown data (or first seen data)against which the model is tested (testing dataset). The goal of crossvalidation is to define a dataset to “test” the model in the trainingphase (i.e., the validation dataset), in order to limit problems likeoverfitting, give an insight on how the model will generalize to anindependent data set (i.e., an unknown dataset, for instance from a realproblem), etc. One round of cross-validation involves partitioning asample of data into complementary subsets, performing the analysis onone subset (called the training set), and validating the analysis on theother subset (called the validation set or testing set). To reducevariability, multiple rounds of cross-validation are performed usingdifferent partitions, and the validation results are averaged over therounds. Cross-validation is important in guarding against testinghypotheses suggested by the data (called “Type III errors”), especiallywhere further samples are hazardous, costly or impossible to collect.The cross-validation method may includes K-fold cross-validation, 2-foldcross-validation, leave-one-out cross-validation. As the name suggests,leave-one-out cross-validation (LOOCV) involves using a singleobservation from the original sample as the validation data, and theremaining observations as the training data. This is repeated such thateach observation in the sample is used once as the validation data. Thisis the same as a K-fold cross-validation with K being equal to thenumber of observations in the original sampling.

The control sample may be a plurality of samples obtained from livercancer patients without cirrhosis, and a plurality of samples obtainedfrom cirrhosis patients without a liver cancer.

The method may further include a process of measuring the level of othertumor markers present in the sample or a control sample, for example inthe separated microvesicles. A tumor marker may be a tumor marker ofliver cancer. The term “tumor marker” used herein refers to a compoundof a moiety specifically found in tumor cells or tissues other thanTMED2 or CD43. The tumor marker may be a protein or nucleic acid, andthe nucleic acid may be miRNA. The tumor marker may be present inmicrovesicles. Thus, measurement of the amount of tumor markers mayinclude a process of directly measuring an amount of one or more tumormarkers that are separated from the microvesicles, or a process ofmeasuring indirectly an amount of one or more tumor markers withoutseparating them from the microvesicles. Measurement may be carried outby detecting signals generated from a detectable label attached to thetumor markers. Separation of the tumor markers may be carried out bycentrifugation, precipitation, salting out, dialysis, filtration,chromatography, or any combination thereof. The chromatography mayinclude affinity chromatography, size-exclusion chromatography, ionexchange chromatography, or any combination thereof. The measuring maybe carried out by ELISA, western blotting, electrophoresis, massspectrometry, spectrometer, or any combination thereof. When the tumormarker is a nucleic acid, the tumor marker may be used to measureamplification products of the nucleic acid. Here, the amplification mayinclude isothermal amplification or thermocycling amplification such asPCR. Nucleic acid markers such as DNA and RNA markers may require use ofa probe relying on fluorescence resonance energy transfer (FRET) pairsin their measuring, and thus, the nucleic acid markers may be measuredin real time at the same time as the amplification thereof. Theamplification may be a real time PCR.

The method may further include a process of measuring the level ofmir-210, mir-346, or a combination thereof that is present in a sampleor a control sample, for example, in the separated microvesicles. Themeasuring may be carried out by separating mir-210, mir-346, or acombination thereof, and measuring directly an amount thereof.Alternatively, the measuring may be carried out by amplifying mir-210,mir-346, or a combination thereof, and measuring an amount thereof.Regarding the amplification, a nucleotide sequence binding to mir-210,mir-346, or a combination thereof, a nucleotide sequence complementaryto mir-210, a nucleotide sequence complementary to mir-346, or anucleotide sequence complementary to a combination of mir-210 andmir-346 may be used as a primer and/or a probe. Such a primer and/or aprobe may be a natural substance, a synthetic substance, or asemi-synthetic substance. The nucleotide sequence may include, forexample, DNA, RNA, DNA-RNA hybrid, PNA, or any combination thereof.

Mir-210 is mature microRNA having a nucleotide sequence of SEQ ID NO: 5,and mir-346 is mature microRNA having a nucleotide sequence of SEQ IDNO: 6.

The nucleotide sequence may be attached to a detectable label. Thedetectable label is defined as described above. The nucleotide sequencemay be fixed to glass support or a solid support. The solid support isdefined as described above.

If the amount of TMED2, CD43, mir-210, mir-346, or any combinationthereof is higher in the subject sample than in a control group sample,the method may further include a process of determining that the subjecthas liver cancer. The control group may be derived from one or morenormal subjects, one or more subjects with a disease other than livercancer, for example, cirrhosis, or a combination thereof. In thisregard, the subject with liver cancer may be distinguished from normalsubjects and subjects with cirrhosis.

According to another aspect of the present invention, there is provideda method of obtaining information for diagnosing liver cancer to asubject, the method including contacting a sample separated from asubject and a substance specifically binding to TMED2, CD43, or acombination thereof wherein the sample comprises microvesicles to whichthe substance can bind; and measuring the amount of the substancecontained in the sample.

The phrases “contacting of the sample separated from the subject to thesubstance specifically binding to the TMED2, the CD43, or a combinationthereof” and the “measuring of the levels of the substance contained inthe sample” are defined as described above.

Hereinafter, the present invention will be described in further detailwith reference to the following examples. These examples are forillustrative purposes only and are not intended to limit the scope ofthe invention.

Example 1 Selection of a Protein Marker Specific to Liver Cancer

The presence of a marker specific to liver cancer contained inblood-derived microvesicles was confirmed using each of the samplesderived from patients with cirrhosis and patients with liver cancer asexplained below.

Blood samples in a range of 8 ml to 10 ml were each derived from 13patients with cirrhosis without liver cancer and 13 patients with livercancer without a cirrhosis, which are examined and confirmed by X-raycomputed tomography (x-ray CT) scan and/or magnetic resonance imaging(MRI) scan, by using BD Vacutainer® Plus plastic whole blood tubes.X-ray CT is a technology that uses compter-processed x-rays to producetomographic images (virtual “slices”) of specific areas of the scannedobject, allowing the user to see what is inside it without cutting itopen. MRI is a medical imaging technique used in radiology toinvestigate the anatomy and function of the body in both health anddisease. Then, the blood samples were separately centrifuged at 1300×gat a temperature of 4° C. for 10 minutes, and plasma was separatedtherefrom. The separated plasma was then stored at a temperature of −80°C. Thereafter, a plasma sample was prepared by thawing the stored plasmafollowed by centrifugation at 3000×g at a temperature of 4° C. for 5minutes so as to use the supernatants, and remove any precipitates.

Three hundred microliters of the each plasma sample were mixed with 30ul of beads (0.8 ug antibodies/bead ul) each coated with anti-CD9antibodies (#MAB1880: R&D Systems), anti-TMED2 antibodies (#sc-376459:Santa Cruz Biotechnology), or anti-CD43 antibodies (#MAB2038: R&DSystems) by using a test tube (Axygen). Then, the mixture was incubatedat room temperature for 4 hours in a Grant Bio-rotator, and the beadswere separated from the mixture by using a magnetic force. Next, theseparated beads were mixed again with 300 ul of PBS, followed byincubation at room temperature for 3 hours as a washing step. Theantibody-coated beads were prepared as follows: The beads were used tocoat protein G on Dynabead M-270 Amine according to a cross-linkingreaction usingNHS/EDC(N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide),and then each antibody was added thereto for the reaction so as to coatthereon by using dimethylpimelimidate. As a result, 2×10⁶ of the beadswere contained in 1 ul of PBS.

After the PBS was removed, 30 ul of NuPAGE LDS sample buffer (LifeTechnologies), which is a lysis buffer containing lithium dodecylsulfate with a pH value of 8.4, were added to the test tube, followed bythermal treating in a heating block at a temperature of 95° C. for 10minutes, thereby lysing the microvesicles. The resulting lysates weresubject to electrolysis followed by western blotting. The resultsdetected in the western blotting were analyzed with luminescence imagesby using ImageQuant LAS 4000 min with rabbit's anti-integrin-β1 as thefirst antibody and HRP-conjugated anti-rabbit antibody as the secondantibody. Integrin-β1 is a marker that is typically present inmicrovesicles.

Based on band intensity values of the integrin-β1 protein compared tothose measured in the microvesicles separated by using the beads coatedwith anti-CD9 antibodies, the band intensity values measured inmicrovesicles separated by using the beads coated with anti-TMED2antibodies or anti-CD43 antibodies were corrected (i.e., normalized).CD9 is a marker that is typically present in microvesicles, and thus,the band intensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-CD9antibodies denotes an amount of total microvesicles contained in asample or an amount of integrin-β1 protein contained in themicrovesicles.

The ROC curve was generated based on the band intensity values of theintegrin-β1 protein measured in the microvesicles separated by using thebeads coated with anti-TMED2 antibodies, anti-CD43 antibodies, or acombination thereof. Table 1 shows the band intensity values of theintegrin-β1 protein measured in the microvesicles separated by using thebeads coated with anti-TMED2 antibodies and the predicted co-variances.

TABLE 1 Actual disease ***Predict- ***Predict_(—) Sample no. *intensitystate noCV noCV.prob 1 69.84 LC C 0.565479944 2 82.40 LC LC 0.48093397 330.40 LC C 0.790880377 4 111.34 LC LC 0.297528356 5 114.67 LC LC0.279052124 6 52.16 LC C 0.677358047 7 88.74 LC LC 0.438367291 8 83.71LC LC 0.472095241 9 138.54 LC LC 0.168706157 10 119.96 LC LC 0.25119354911 162.62 LC LC 0.095681062 12 39.91 LC C 0.745168058 13 105.75 LC LC0.330064731 14 76.06 C C 0.523779232 15 62.99 C C 0.610334267 16 90.23 CLC 0.428470602 17 89.19 C LC 0.435372812 18 75.37 C C 0.528432409 1928.89 C C 0.797555246 20 78.83 C C 0.505065667 21 9.29 C C 0.87003203 2260.30 C C 0.627493535 23 86.16 C LC 0.455615913 24 62.26 C C 0.61501994925 76.02 C C 0.524049102 26 81.61 C LC 0.486270325 *‘intensity” refersto band intensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-TMED2antibodies. ** Predict-noCV refers to a predicted normalized covariance.******Predict_noCV.prob refers to a predicted normalized covarianceproablility. “LC” refers to liver cancer and “C” refers to cirrhosis.

The intensity data shown in Table 1 is analyzed by using leave-one-outcross-validation method. The logistic regression model is used in theanalysis by using leave-one-out cross-validation method.

Table 2 shows test results using a value obtained the analysis by usingleave-one-out cross-validation method, that is, 81.61 TMED (normalized).That is, when the intensity value is equal to or greater than 81.61, thesubject is determined to be a liver cancer patient, and when theintensity value is less than 81.61, the subject is determined to be acirrhosis patient. The results for subjects diagnosed with cirrhosis orliver cancer based on the band intensity values of the integrin-β1protein measured in the microvesicles separated by using the beadscoated with anti-TMED2 antibodies are shown in Table 2 below. Theprediction results was tested by using a logistic regression methodusing a determination function=2.153-0.027*TMED2 which is a probabilityof a patient to be a liver cancer patient, wherein TMED2 refers to bandintensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-TMED2antibodies, and the subject is determined to be a liver cancer patientwhen the determination function value is equal to or greater than 0.5,and the subject is determined to be a cirrhosis patient when thedetermination function value is less than 0.5. The results denote anarea under the curve (AUC) of 0.693, sensitivity of 0.692, andspecificity of 0.692. FIG. 1 is a graph showing a ROC curve to determinepresence of cirrhosis or liver cancer in the sample based on the bandintensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-TMED2antibodies.

TABLE 2 Actual cirrhosis Actual liver cancer Total *Predicted cirrhosis9 4 13 *Predicted liver cancer 4 9 13 total 13 13 26 *“Predicted”cirrhosis or liver cancer is determined by using the value 81.61obtained by the leave-one-out cross-validation method.

Table 3 shows the band intensity values of the integrin-β1 proteinmeasured in the microvesicles separated by using the beads coated withanti-CD43 antibodies and the predicted co-variances.

TABLE 3 Actual disease ***Predict- ***Predict_(—) Sample no. *intensitystate noCV noCV.prob 1 68.47 LC LC 0.457221 2 0.52 LC C 0.576041 3 16.73LC C 0.547975 4 17.78 LC C 0.546144 5 17.86 LC C 0.546005 6 24.84 LC C0.533807 7 95.03 LC LC 0.411347 8 42.01 LC C 0.503657 9 36.38 LC C0.513557 10 148.09 LC LC 0.324819 11 144.75 LC LC 0.329994 12 20.99 LC C0.540541 13 9.62 LC C 0.560333 14 88.62 C LC 0.422309 15 21.32 C C0.539964 16 38.29 C C 0.510199 17 27.86 C C 0.528515 18 15.62 C C0.549909 19 42.29 C C 0.503165 20 77.70 C LC 0.441156 21 16.52 C C0.548341 22 74.71 C LC 0.446348 23 17.02 C C 0.547469 24 43.59 C C0.500878 25 26.10 C C 0.5316 26 22.04 C C 0.538706 *‘intensity” refersto band intensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-CD43antibodies. ** Predict-noCV refers to a predicted normalizedcovariance.. ******Predict_noCV.prob refers to a predicted normalizedcovariance probability. “LC” refers to liver cancer and “C” refers tocirrhosis.

The intensity data shown in Table 3 is analyzed by using leave-one-outcross-validation method. The logistic regression model is used in theanalysis by using leave-one-out cross-validation method.

Table 4 shows test results using a value obtained the analysis by usingleave-one-out cross-validation method, that is, 68.47 CD43 (normalized).That is, when the intensity value is equal to or greater than 68.47, thesubject is determined to be a liver cancer patient, and when theintensity value is less than 68.47, the subject is determined to be acirrhosis patient. The results for subjects diagnosed with cirrhosis orliver cancer based on the band intensity values of the integrin-β1protein measured in the microvesicles separated by using the beadscoated with anti-CD 43 antibodies are shown in Table 4 below. Theprediction results was tested by using a logistic regression methodusing a determination function=0.31-0.007*CD43 which is a probability ofa patient to be a liver cancer patient, wherein CD43 refers to bandintensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-CD43antibodies, and the subject is determined to be a liver cancer patientwhen the determination function value is equal to or greater than 0.5,and the subject is determined to be a cirrhosis patient when thedetermination function value is less than 0.5. The results denote AUC of0. 0.538, sensitivity of 0.308, and specificity of 0.769. FIG. 2 is agraph showing a ROC curve to determine the presence of cirrhosis orliver cancer in the sample based on the band intensity values of theintegrin-β1 protein measured in the microvesicles separated by using thebeads coated with anti-CD 43 antibodies.

TABLE 4 Actual cirrhosis Actual liver cancer Predicted cirrhosis 10 9 19Predicted liver cancer 3 4 7 Total 13 13 26 * “Predicted” cirrhosis orliver cancer is determined by using the value 68.47 obtained by theleave-one-out cross-validation method.

Table 5 shows the band intensity values of the integrin-β1 proteinmeasured in the microvesicles separated by using the beads coated withanti-TMED2 antibodies and anti-CD43 antibodies and the predictedco-variances.

TABLE 5 Actual Sample *TMED2 *CD43 disease ***Predict- ***Predict_(—)no. intensity intensity state noCV noCV.prob 1 69.84 68.47 LC C 0.5716542 82.4 0.52 LC LC 0.471643 3 30.4 16.73 LC C 0.789743 4 111.34 17.78 LCLC 0.290976 5 114.67 17.86 LC LC 0.272554 6 52.16 24.84 LC C 0.675877 788.74 95.03 LC LC 0.448743 8 83.71 42.01 LC LC 0.471603 9 138.54 36.38LC LC 0.165346 10 119.96 148.09 LC LC 0.266111 11 162.62 144.75 LC LC0.101132 12 39.91 20.99 LC C 0.743987 13 105.75 9.62 LC LC 0.321978 1476.06 88.62 C C 0.533879 15 62.99 21.32 C C 0.607186 16 90.23 38.29 C LC0.4267 17 89.19 27.86 C LC 0.431474 18 75.37 15.62 C C 0.522938 19 28.8942.29 C C 0.800062 20 78.83 77.7 C C 0.51263 21 9.29 16.52 C C 0.8699922 60.3 74.71 C C 0.635303 23 86.16 17.02 C LC 0.449609 24 62.26 43.59 CC 0.616479 25 76.02 26.1 C C 0.520749 26 81.61 22.04 C Liver 0.481653cancer *“TMED2 intensity” and “CD43 intensity” refers to band intensityvalues of the integrin-β1 protein measured in the microvesiclesseparated by using the beads coated with anti-TMED2 antibodies andanti-CD43 antibodies, respectively. ** Predict-noCV refers to apredicted normalized covariance. ******Predict_noCV.prob refers to apredicted normalized covariance probability. “LC” refers to liver cancerand “C” refers to cirrhosis.

The intensity data shown in Table 5 is analyzed by using leave-one-outcross-validation method. The logistic regression model is used in theanalysis by using leave-one-out cross-validation method.

Table 6 shows test results that the prediction was conducted by using alogistic regression method using a determinationfunction=0.31-0.007*CD43 which is a probability of a patient to be aliver cancer patient, wherein CD43 refers to band intensity values ofthe integrin-β1 protein measured in the microvesicles separated by usingthe beads coated with anti-TMED2 antibodies and anti-CD43 antibodies,and the subject is determined to be a liver cancer patient when thedetermination function value is equal to or greater than 0.5, and thesubject is determined to be a cirrhosis patient when the determinationfunction value is less than 0.5. The results of subjects diagnosed withcirrhosis or liver cancer based on the band intensity values of theintegrin-β1 protein measured in the microvesicles separated by using thebeads coated with anti-TMED2 antibodies and anti-CD43 antibodies areshown in Table 6 below. The results denote AUC of 0.692, sensitivity of0.692, and specificity of 0.692. FIG. 3 is a graph showing a ROC curveto determine presence of cirrhosis or liver cancer in the sample basedon the band intensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-TMED2antibodies and anti-CD43 antibodies.

TABLE 6 Actual cirrhosis Actual liver cancer Total Predicted cirrhosis 94 13 Predicted liver cancer 4 9 13 total 13 13 26 * “Predicted”cirrhosis or liver cancer is determined by using the probability value0.5 obtained by a determination function.

The above results demonstrate it is possible to distinguish liver cancersamples from cirrhosis samples by using the TMED2 and CD43 as tumormarkers.

Example 2 Selection of Liver Cancer-Specific Protein and miRNA Marker

The presence of a liver cancer-specific protein and a miRNA marker inblood-derived microvesicles was confirmed using samples taken from apatient with cirrhosis and a patient with liver cancer.

Blood samples in a range of 8 ml to 10 ml were each derived from 13patients with cirrhosis and 13 patients with liver cancer by using BDVacutainer® Plus plastic whole blood tubes. Then, the blood samples wereseparately centrifuged at 1300×g at a temperature of 4° C. for 10minutes, and accordingly plasma was separated therefrom. The separatedplasma was then stored at a temperature of −80° C. A plasma sample wasprepared by thawing the stored plasma followed by centrifuged at 3000×gat a temperature of 4° C. for 5 minutes so as to use the supernatants,and remove any precipitates.

Three hundred microliters of the plasma sample were mixed with 30 ul ofbeads (0.8 ug antibodies/bead ul) each coated with anti-CD9 antibodies(R&D Systems), anti-TMED2 antibodies (Santa Cruz Biotechnology), oranti-CD43 antibodies (R&D Systems) by using a test tube (Axygen). Then,the mixture was incubated at room temperature for 4 hours in Grant Biorotator, and the beads were separated the mixture by using magneticforces. Next, the separated beads were mixed again with 300 ul of PBS,followed by incubation at room temperature for 3 hours, as a washingstep. The antibody-coated beads were prepared as follows: The beads wereused to coat protein G on Dynabead M-270 Amine according to across-linking reaction using NHS/EDC, and then each antibody was addedthereto for the reaction so as to coat thereon by usingdimethylpimelimidate. As a result, 2×10⁶ of the beads were contained in1 ul of PBS.

After the PBS was removed, miRNA was extracted by using a miRNeasy kitaccording to the manufacturer's instructions. Then, cDNA was synthesizedtherefrom using a Universal cDNA synthesis kit (Exiqon), and real timePCR was performed thereto by using a SYBR Green master mix kit (Exiqon)according to the manufacturer's instructions. The primer used herein wasprovided by the manufacturer.

Based on the absorbance value measured at a wavelength in a range fromabout 483 nm to about 533 nm in RT-PCR, an ROC curve analysis wasperformed in view of a combination of the absorbance value of mir-210measured in the microvesicles separated by using the beads coated withthe anti-TMED2 (hereinafter “TMED2_hsa.miR.210”) and the absorbancevalue of mir-346 measured in the microvesicles separated by using thebeads coated with the anti-CD43(hereinafter “CD43_hsa.miR.346”).

Table 7 shows the band intensity values of the integrin-β1 proteinmeasured in the microvesicles separated by using the beads coated withanti-TMED2 antibodies and anti-CD43 antibodies, TMED2_hsa.miR.210,CD43_hsa.miR.346, and the predicted co-variances.

TABLE 7 Actual *TMED2 *CD43 disease ***Predict- no. intensity intensityCD43_hsa.miR.346 TMED2_hsa.miR.210 state noCV ***Predict_noCV.prob 169.84 68.47 29 26 LC LC 0.083348521 2 82.4 0.52 29 27.2 LC LC0.053495722 3 30.4 16.73 30 26.4 LC LC 0.295590048 4 111.34 17.78 3032.5 LC C 0.610961166 5 114.67 17.86 29.9 25.7 LC LC 0.019698477 6 52.1624.84 24.5 31.1 LC LC 0.074329151 7 88.74 95.03 27.4 30 LC LC0.207337793 8 83.71 42.01 29.5 30 LC LC 0.421408953 9 138.54 36.38 24.930 LC LC 0.004583781 10 119.96 148.09 28.2 27.8 LC LC 0.08253975 11162.62 144.75 31 27.3 LC LC 0.117406475 12 39.91 20.99 30.2 30.9 LC C0.859563171 13 105.75 9.62 30.1 30 LC LC 0.279624467 14 76.06 88.62 3030 C C 0.700420143 15 62.99 21.32 26.2 34.2 C C 0.561792216 16 90.2338.29 28.8 34 C C 0.801398191 17 89.19 27.86 31 33.9 C C 0.944395352 1875.37 15.62 34.1 37.1 C C 0.999337077 19 28.89 42.29 34.9 33.5 C C0.999398551 20 78.83 77.7 30 30.3 C C 0.693412076 21 9.29 16.52 33 30 CC 0.984624826 22 60.3 74.71 34.6 30 C C 0.988915782 23 86.16 17.02 3526.1 C C 0.708787136 24 62.26 43.59 34.4 31.5 C C 0.992062106 25 76.0226.1 29.9 27.7 C LC 0.196926618 26 81.61 22.04 28.8 30.4 C LC0.318642451 *“TMED2 intensity” and “CD43 intensity” refers to bandintensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-TMED2antibodies and anti-CD43 antibodies. ***Predict-noCV refers to apredicted normalized covariance. ***Predict_noCV.prob refers to apredicted normalized covariance probability. “LC” refers to liver cancerand “C” refers to cirrhosis.

The intensity data shown in Table 7 is analyzed by using leave-one-outcross-validation method. The logistic regression model is used in theanalysis by using leave-one-out cross-validation method.

Table 8 shows test results that the prediction was conducted by using alogistic regression method using a determinationfunction=−38.162+0.012*CD43-0.03*TMED2+0.616*TMED2_hsa.miR.210+0.725*CD43_hsa.miR.346which is a probability of a patient to be a liver cancer patient,wherein CD43 refers to band intensity values of the integrin-β1 proteinmeasured in the microvesicles separated by using the beads coated withanti-TMED2 antibodies and anti-CD43 antibodies, and the subject isdetermined to be a liver cancer patient when the determination functionvalue is equal to or greater than 0.5, and the subject is determined tobe a cirrhosis patient when the determination function value is lessthan 0.5. The results for subjects diagnosed with cirrhosis or livercancer based on such band intensity and absorbance value above are shownin Table 8 below. The results denote an area under the curve (AUC) of0.85, sensitivity of 0.85, and specificity of 0.85. FIG. 4 is a graphshowing a ROC curve to determine the presence of cirrhosis or livercancer in the sample based on the combined values of mir-210 withrespect to the microvesicles that were separately by using the beadscoated with anti-TMED2 and mir-346 with respect to the microvesiclesthat were separately by using the beads coated with anti-CD43 and bandintensity values of the integrin-β1 protein measured in themicrovesicles separated by using the beads coated with anti-TMED2antibodies and anti-CD43 antibodies, respectively.

TABLE 8 Actual cirrhosis Actual liver cancer total Predicted cirrhosis11 2 13 Predicted liver cancer 2 11 13 total 13 13 26 * “Predicted”cirrhosis or liver cancer is determined by using the probability value0.5 obtained by a determination function.

The above results demonstrate it is possible to distinguish a sample ofliver cancer from a sample of cirrhosis by using the TMED2 and CD43 astumor markers as well mir-210 and mir-346 expression values.

As described above, according to the one or more of the aboveembodiments of the present invention, a composition for diagnosing livercancer in a subject may have high efficiency. According to one or moreof the above embodiments of the present invention, a method ofdiagnosing liver cancer in a subject may be used to efficiently diagnoseliver cancer in a subject. According to one or more of the aboveembodiments of the present invention, a method of obtaining informationfor diagnosing liver cancer in a subject may have high efficiency.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A composition for diagnosing liver cancer in asubject, wherein the composition comprises a substance that specificallybinds to transmembrane emp24 domain trafficking protein 2 (TMED2),cluster of differentiation 43 (CD43), or a combination thereof.
 2. Thecomposition according to claim 1, wherein the TMED2, CD43, orcombination thereof is present on the surface of microvesicles separatedfrom a living body.
 3. The composition according to claim 1, furthercomprising a reagent for measuring expression levels of mir-210,mir-346, or a combination thereof.
 4. The composition according to claim3, wherein the reagent binds to mir-210, mir-346, a combination thereof,or to nucleotide sequences complementary to mir-210, mir-346, or acombination thereof.
 5. The composition according to claim 1, whereinthe substance is an antibody or an antigen-binding fragment thereof, aligand, a substrate, an inhibitor, an agonist, an antagonist, aco-factor, or any combination thereof.
 6. The composition according toclaim 1, wherein the substance is attached to a detectable label.
 7. Amethod of diagnosing liver cancer in a subject, the method comprising:contacting a sample from a subject with a substance that specificallybinds to transmembrane emp24 domain trafficking protein 2 (TMED2),cluster of differentiation 43 (CD43), or any combination thereof on thesurface of a microvesicle in the sample, measuring the level of thesubstance bound to microvesicles in the sample; measuring the level ofthe substance bound to microvesicles in the control sample; anddetermining that the subject has a liver cancer or an increasedlikelihood of developing a liver cancer when the level of the substancebound to microvesicles in the sample is equal or greater than athreshold level, or when the level of the substance bound tomicrovesicles in the sample is greater than the highest level amonglevels obtained from the plurality of samples obtained from cirrhosispatients, wherein the control sample includes a plurality of samplesobtained from liver cancer patients and a plurality of samples obtainedfrom cirrhosis patients, and the threshold level is a value such thatthe probability of a sample to be a liver cancer patient is equal to orgreater than 50% to 100% and the probability of a sample to be a livercancer patient is obtained by analyzing the measured level with astatistic model.
 8. The method according to claim 7, wherein statisticmodel is a logistic regression model.
 9. The method according to claim8, wherein the control sample is a plurality of samples obtained fromliver cancer patients without cirrhosis, and a plurality of samplesobtained from cirrhosis patients without a liver cancer.
 10. The methodaccording to claim 7, further comprising separating the microvesiclesfrom the sample after contacting the sample with the substance.
 11. Themethod according to claim 10, wherein separating the microvesicles fromthe sample comprises centrifugation, use of a magnetic force, bindingthe microvesicles to a solid support, or any combination thereof. 12.The method according to claim 11, wherein the substance is attached to adetectable label.
 13. The method according to claim 7, furthercomprising: measuring the level of one or more tumor markers other thanTMED2 or CD43 in the sample.
 14. The method according to claim 13,further comprising: measuring levels of mir-210, mir-346, or acombination thereof present in the sample; and measuring the levels ofmir-210, mir-346, or a combination thereof present in the controlsample.
 15. The method according to claim 13, wherein the reagent isattached to a detectable label.
 16. A composition comprising amicrovesicle comprising TMED2, CD43, or both on the surface of themicrovesicle, a substance that specifically binds TMED2, CD43, or bothbound to the microvesicle, and a detectable label bound to the substancethat specifically binds TMED2, CD43, or both.